Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions

The detection of physical interaction between two or more molecules of interest can be facilitated if the act of association between the interactive partners leads to the production of a readily observed biological or physical readout. Many interacting molecule pairs (X, Y) can be made to induce such a readout if X and Y are each fused to defined protein elements with desired properties. For example, in the yeast forward two-hybrid system, X is synthesized as a translational fusion to a DNA-binding domain (DBD), Y is synthesized as a fusion to a transcriptional activation domain (AD), and coexpression of DBD-X and AD-Y induces transcription of easily scored responsive reporters. Other approaches use paradigms based on the artifical production of two, hybrid, molecules, but substitute a variety of readouts including the repression of transcription, activation of signal transduction pathways, or reconstitution of a disrupted enzymatic activity. In this article, we summarize a number of two-hybrid-based approaches, and detail the use of the forward yeast two-hybrid system in a screen to identify novel interacting partners for a protein of interest. (C) 2001 Academic Press.